Ribosome-mediated incorporation of peptides and peptidomimetics
Inventors
Hecht, Sidney • DEDKOVA, Larisa (Liza) • Maini, Rumit • ROY CHOWDHURY, Sandipan • Paul, Rakesh
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Assignees
Arizona State University ASU • Arizona State University Downtown Phoenix campus
Member
Arizona State UniversityArizona State University (ASU) is a comprehensive public research university known for its commitment to inclusivity, excellence, and societal impact. ASU is dedicated to advancing research-based solutions for global challenges, fostering strong leadership, and providing quality education accessible to all learners. The university is recognized for its innovative approach to education and its efforts in sustainability, research, and community service. ASU exemplifies a new prototype for the American public research university, focusing on excellence, broad access to quality education, and meaningful societal impact.
Arizona State University (ASU) is a comprehensive public research university known for its commitment to inclusivity, excellence, and societal impact. ASU is dedicated to advancing research-based solutions for global challenges, fostering strong leadership, and providing quality education accessible to all learners. The university is recognized for its innovative approach to education and its efforts in sustainability, research, and community service. ASU exemplifies a new prototype for the American public research university, focusing on excellence, broad access to quality education, and meaningful societal impact.
Publication Number
US-10745705-B2
Publication Date
2020-08-18
Expiration Date
2036-01-22
Abstract
Modified ribosomes that were selected using a dipeptidyl-puromycin aminonucleoside are used to mediate site-specific incorporation of one or more peptides and peptidomimetics into protein in a cell free translation system. In addition, new fluorescent dipeptidomimetics have been synthesized and incorporated into proteins, as well as modified proteins containing one or more non-naturally occurring dipeptides.
Core Innovation
This invention relates to the use of genetically modified ribosomes, specifically those with a 23S rRNA sequence modification, to mediate the site-specific incorporation of peptides and peptidomimetics into proteins within a cell-free translation system. These modified ribosomes were selected using a dipeptidyl-puromycin aminonucleoside derivative. For the first time, it is demonstrated that such modified ribosomes can facilitate the incorporation of multiple dipeptides and dipeptidomimetics into a protein using a single codon in the mRNA, enabling the synthesis of proteins with non-naturally occurring dipeptides and fluorescent dipeptidomimetics.
The problem addressed is the long-standing challenge in peptide and protein engineering: achieving the incorporation of non-standard chemical structures, such as modified peptides or peptidomimetics, into proteins to alter molecular properties like stability or biological activity. Traditional ribosomes have very limited capability to accept these modifications, restricting the diversity of engineered proteins and hindering the development of novel drug-like compounds from peptides or peptidomimetics.
Embodiments of the invention include methods for selecting and using modified ribosomes capable of incorporating a broad range of dipeptides and dipeptidomimetics using suppressor tRNAs acylated with these substrates. The invention encompasses in vitro translation systems and protocols for producing proteins containing these non-naturally occurring components, as well as the preparation and application of new fluorescent dipeptidomimetics and fluorescent proteins incorporating them.
Claims Coverage
The independent claims cover two main inventive features relating to in vitro translation systems and methods for protein modification using tRNAs with modified dipeptidomimetics.
In vitro translation system comprising a tRNA with modified dipeptidomimetic
An in vitro translation system is provided that includes materials sufficient to effect translation from a messenger RNA (mRNA), wherein the system includes a transfer RNA (tRNA) that is acylated or otherwise modified with a dipeptidomimetic.
Method for producing a modified peptide, polypeptide, or protein using a tRNA with modified dipeptidomimetic
A method is disclosed for producing a modified peptide, polypeptide, or protein. The method comprises providing to an in vitro translation system a transfer RNA (tRNA) that is loaded with a modified dipeptidomimetic.
The claims focus on systems and methods for incorporating modified dipeptidomimetics into proteins using tRNAs in cell-free translation, enabling the construction of modified proteins.
Stated Advantages
The invention enables the site-specific incorporation of dipeptides and dipeptidomimetics into proteins using a single codon in a cell-free translation system, something not achieved with wild-type ribosomes.
It allows for the creation of proteins containing non-naturally occurring dipeptides and new fluorescent dipeptidomimetics within a controlled in vitro environment.
Modified proteins generated with cyclic peptidomimetics exhibit higher proteolytic stability compared to wild-type proteins.
Documented Applications
Preparation of modified proteins, polypeptides, or peptides containing one or more site-specific non-naturally occurring dipeptides or dipeptidomimetics.
Synthesis of artificial fluorescent proteins embedding novel fluorescent dipeptidomimetics as analogues of natural fluorophores.
Production of modified enzymes (e.g., DHFR) with enhanced stability or new functional properties by site-specific incorporation of cyclic peptidomimetics.
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